Both Alpha- and Beta-Rhizobia Occupy the Root Nodules of Vachellia karroo in South Africa

Abstract

Vachellia karroo (formerly Acacia karroo) is a wide-spread legume species indigenous to southern Africa. Little is known regarding the identity or diversity of rhizobia that associate with this plant in its native range in South Africa. The aims of this study were therefore: (i) to gather a collection of rhizobia associated with V. karroo from a wide range of geographic locations and biomes; (ii) to identify the isolates and infer their evolutionary relationships with known rhizobia; (iii) to confirm their nodulation abilities by using them in inoculation assays to induce nodules under glasshouse conditions. To achieve these aims, soil samples were collected from 28 locations in seven biomes throughout South Africa, which were then used to grow V. karroo seedlings under nitrogen-free conditions. The resulting 88 bacterial isolates were identified to genus-level using 16S rRNA sequence analysis and to putative species-level using recA-based phylogenetic analyses. Our results showed that the rhizobial isolates represented members of several genera of Alphaproteobacteria (Bradyrhizobium, Ensifer, Mesorhizobium, and Rhizobium), as well as Paraburkholderia from the Betaproteobacteria. Our study therefore greatly increases the known number of Paraburkholderia isolates which can associate with this southern African mimosoid host. We also show for the first time that members of this genus can associate with legumes, not only in the Fynbos biome, but also in the Albany Thicket and Succulent Karoo biomes. Twenty-six putative species were delineated among the 88 isolates, many of which appeared to be new to Science with other likely being conspecific or closely related to E. alkalisoli, M. abyssinicae, M. shonense, and P. tropica. We encountered only a single isolate of Bradyrhizobium, which is in contrast to the dominant association of this genus with Australian Acacia. V. karroo also associates with diverse genera in the Grassland biome where it is quite invasive and involved in bush encroachment. Our findings therefore suggest that V. karroo is a promiscuous host capable of forming effective nodules with both alpha- and beta-rhizobia, which could be a driving force behind the ecological success of this tree species.

Keywords: Acacia karroo; Bradyrhizobium; Paraburkholderia; South Africa; Vachellia karroo; alpha-rhizobia; beta-rhizobia.

FIGURE 1

(A) Biome map of South Africa, Lesotho and Swaziland (as in Driver et al., 2005; from the national Spatial Biodiversity Assessment of 2004, courtesy of SANBI). The nine biomes are coloured according to the key and black dots indicate the sampling localities. (B) Summary of the putative species encountered in this study. (C) The core of this diagram lists the seven sampled biomes (colour-coded as in A). The second track indicates the genera isolated from each biome (with alpha-rhizobia appearing in shades of green while beta-rhizobia appear in blue), while the third track shows the number of isolates sampled from each genus for that specific biome relative to the number of recA phylogeny-based putative species they formed part of. The outer track shows the number of unique recA phylogeny-based putative species recovered for a specific biome for each rhizobial genus. We were unable to amplify the recA gene sequence for the Mesorhizobium isolate recovered from the Albany Thicket which is why it is indicated with an asterisk, as it could not be assigned to a species.

FIGURE 2

A maximum-likelihood phylogeny of the recA locus for the beta-rhizobial genus Paraburkholderia. Isolates from this study are listed in red, while type strains of species appear in bold. The delineated Vachellia karroo-associated lineages (VK-13 to VK-22) appear in blue. In order to minimize the phylogeny some lineages (which do not contain any isolates from this study), have been collapsed. The expanded tree, containing information for the host or source and geographic origin of each isolate, appears as Supplementary Figure S3. GenBank accession numbers for all the isolates included in this phylogeny are listed in Supplementary Table S3. Nodes with ≥50% are indicated, while the scale bar shows the number of nucleotide substitutions per site.